|
| Status |
Public on Apr 08, 2009 |
| Title |
Lesion vs mycelia/rice biological rep 3.2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
36 hpi infected rice sheath
|
| Organisms |
Oryza sativa; Pyricularia oryzae |
| Characteristics |
Biotrophic invasive hyphae in rice sheath epidermal cells
|
| Biomaterial provider |
B. Valent Lab. Kansas State University
|
| Treatment protocol |
Rice leaf sheath pieces were placed in wire supports in order to avoid contact with the wet paper, and to hold the pieces horizontally flat for even inoculum distribution over the mid-vein. Spore suspension (1 X 105 spores/mL in gelatin solution) was injected in one end of the leaf sheath using a 1 mL pipet.
|
| Growth protocol |
Inoculated tissues were kept in high humidity at room temperature for 36 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol method as specified by the manufacturers.
|
| Label |
cy5
|
| Label protocol |
Total RNA was labeled using a linear amplification kit (Agilent Technologies). Five hundred nanograms were one-round amplified. Normal cRNA yields were 10-15 ug. The cRNA was labeled according to manufacturers specifications. Fluorescent cRNA was purified, quantified using the NanoDrop Spectrophotometer, and 1 µg of probe was hybridized to M. oryzae microarray slides.
|
| |
|
| Channel 2 |
| Source name |
20% mycelial RNA mixed with 80% mock-inoculated rice RNA
|
| Organisms |
Oryza sativa; Pyricularia oryzae |
| Characteristics |
Fungal and plant RNAs mixed in a similar proportion as in the infected samples
|
| Biomaterial provider |
B. Valent Lab. Kansas State University
|
| Treatment protocol |
Sheaths inoculated with gelatin solution. An approximately 1 cm square piece of agar containing fungus was excised from the surface of an oatmeal agar plate and blended in 25 ml of 3,3,3 liquid medium (3g/L of glucose, 3g/L of casamino acids, and 3g/L of yeast extract). The blended mycelium was mixed with 225 ml of fresh medium in 500 ml flasks.
|
| Growth protocol |
Inoculated sheath tissues were incubated in high humidity at room temperature for 36 hours. The fungal culture was incubated at 24°C under continuous rotation at 120 RPM. Mycelium was collected by filtration after 24 h of incubation, and the blending treatment was repeated for 3 rounds of growth
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol method as specified by the manufacturers
|
| Label |
cy3
|
| Label protocol |
Total RNA was labeled using a linear amplification kit (Agilent Technologies). Five hundred nanograms were one-round amplified. Normal cRNA yields were 10-15 ug. The cRNA was labeled according to manufacturers specifications. Fluorescent cRNA was purified, quantified using the NanoDrop Spectrophotometer, and 1 µg of probe was hybridized to M. oryzae microarray slides.
|
| |
|
| |
| Hybridization protocol |
The labeled samples (1ug each cy3 and cy5 labeled cRNA) were defragmented by the addition of 25X Agilent Defragmentation buffer and incubated for 30 min, 60°C. The sample was adjusted to a final volume of 450 µl with formamide-containing hybridization buffer (Hughes et al., 2001) and then added to the microarray slides. Slides were incubated for 18 hours with continuous rotation at 40°C. After hybridization, slides were washed in 6X SSPE, 0.005% sarcosyl for 1 min, in 0.06X SSPE for 30 sec, in water for 30 sec and then air dried.
|
| Scan protocol |
Slides were scanned on an Agilent G2565BA DNA microarray scanner, and the TIFF images extracted using the Agilent Feature Extraction software (version 8.5). The resultant .xml and .jpeg files were imported into Rosetta Resolver.
|
| Description |
This experiment was designed to compare gene expression
by biotrophic invasive hyphae in planta to gene expression
by mycelium in nutrient medium
|
| Data processing |
Data from individual biological replicates and also the combined data were analyzed. The data was then plotted as an intensity scatter plot (background subtracted/dye normalized cy3channel vs background subtracted/dye normalized cy5 channel). “Signature sequences” (features which were 1.5 fold differentially regulated, P-values <0.01) were identified by Resolver, and exported to Excel.
|
| |
|
| Submission date |
Jul 12, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Barbara Valent |
| E-mail(s) |
bvalent@ksu.edu
|
| Phone |
785-532-2336
|
| Fax |
785-532-5692
|
| Organization name |
Kansas State University
|
| Department |
Department of Plant Pathology
|
| Street address |
4024 Throckmorton Plant Sciences
|
| City |
Manhattan |
| State/province |
KS |
| ZIP/Postal code |
66506-5502 |
| Country |
USA |
| |
|
| Platform ID |
GPL892 |
| Series (2) |
| GSE8518 |
Rice Gene Expression During Biotrophic Invasion by Magnaporthe oryzae (Agilent Array G4138A) |
| GSE8670 |
Analysis of the Interaction Transcriptome During Biotrophic Invasion of Rice by the Blast Fungus, Magnaporthe oryzae |
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